Gene expression analysis is carried out by extracting mRNA from cells, preparing a complementary strand thereof; i.e., cDNA, increasing its copy number via PCR, and capturing the target at a relevant probe position using a DNA probe array (DNA chip) to detect it on the basis of fluorescence. PCR amplification or a method involving the use of a DNA chip, however, is disadvantageous in terms of the low accuracy of quantitative analysis. Accordingly, development of a method for analyzing gene expression profiles with high accuracy has been awaited. Along with the completion of human genome analysis, the demand for quantitative gene expression analysis is increasing. In recent years, however, a method comprising extracting mRNA from a single cell and quantitatively analyzing it has been needed. An example of a method of analysis with high quantitative efficiency is quantitative PCR. This method of quantitative analysis comprises preparing a standard sample with the same DNA sequence as the target, performing PCR amplification under the same conditions, and observing and comparing the progression of amplification using fluorescent probes. When the target is a single cell, the number of existing mRNA is small, and quantitative analysis is difficult to conduct. When expression of a plurality of genes is to be quantitatively analyzed, samples are divided and independently subjected to quantitative analysis. When the number of target genes is large and expression levels of some genes are low, accordingly, analysis may be sometimes impossible to conduct as a result of sample division.
Under the above circumstances, the inventors developed a method comprising converting all mRNA into cDNA, preparing a cDNA library retained on beads (i.e., a cDNA population including all cDNA), and using it for quantitative analysis. They demonstrated that errors in determination of genes expressed at very low levels caused by sample division could be eliminated with the repeated use of a cDNA library and expression levels of a plurality of genes contained in a single cell could be accurately determined (Non-Patent Document 1: Nature Method, Vol. 6, No. 7, 2009, pp. 503-506).
In the method described above, all the processes, including mixing of a reaction reagent with a sample-containing solution and purification, are performed manually. From the viewpoint of fractionation accuracy and solvent evaporation, the volume of the reaction solution is limited to at least several hundreds nanoliters to microliters. This necessitates the use of a reagent at an adequate concentration even when a single cell is to be analyzed. Accordingly, the amount of a reaction reagent (i.e., the number of moles) increases in proportion to the reaction volume, which in turn increases reagent cost. When assay of numerous cells is necessary in order to achieve statistically significant data, the reagent cost would be very high. Accordingly, a method that can he carried out with a reduced reaction volume and solves problems in fractionation accuracy and evaporation has been desired.
In order to overcome the aforementioned problems, a method involving the use of a device comprising small flow channels referred to as “microfluidics” in combination was adopted in the past. An example in which microfluidics is employed for single cell analysis is described in Non-Patent Document 2 (Proceedings of the National Academy of Sciences, Vol. 108, No. 34, 2011, pp. 13999-14004). FIG. 1 of Non-Patent Document 2 shows the constitution of a device in which a chip is composed of 300 unit structures, so that 300 cells can be simultaneously treated. 3×50 such unit structures are arranged on a chip, a unit structure is horizontally long, a sample solution flows in a longitudinal direction, and the reaction is successively carried out while the sample solution continues to flow. The reaction volume is 10 nL at the time of reverse transcription and it is 50 nL at the time of PCR. That is, the reaction volume is reduced.
In order to simultaneously treat a large number of cells, in addition, it is necessary to simultaneously subject many cells to gene expression analysis in a cost-effective manner. To this end, a method in which a cDNA library is constructed with the use of porous membranes instead of beads is described in Patent Document 1 (JP 2009-276883 A). This method involves the use of a device that obtains two-dimensional distribution of gene expression and implements gene expression analysis of many cells. When genes in a single cell are subjected to expression analysis with the use of such device, it is not necessary to isolate cells. Thus, mRNA can be directly extracted from cells originating from biological tissue sections and can be subjected to gene expression analysis. In order to increase the number of genes that can be analyzed, however, it was necessary to perform fluorescence assay or chemiluminesence assay repeatedly, in proportion to the number of genes assayed.